Receptor specific transepithelial transport of therapeutics

ABSTRACT

The present invention relates in general to methods and products for initiating an immune response against an antigen, and in particular relates to transepithelial delivery of antigens to provoke tolerance and immunity. The present invention further relates to methods and products for the transepithelial delivery of therapeutics. In particular, the invention relates to methods and compositions for the delivery of therapeutics conjugated to a FcRn binding partner to intestinal epithelium, mucosal epithelium and epithelium of the lung. The present invention further relates to the synthesis, preparation and use of the FcRn binding partner conjugates as, or in, pharmaceutical compositions for oral systemic delivery of drugs and vaccines.

[0001] This application is a continuation-in-part of U.S. Ser. No.08/899,856 filed Jul. 24, 1997 which is a continuation-in-part of U.S.Ser. No. 08/578,171 filed on Dec. 29, 1995, now pending, which is acontinuation-in-part of U.S. Ser. No. 08/374,159 filed on Jan. 17, 1995,now pending.

[0002] The work described herein was supported, in part, by NationalInstitutes of Health Grant Nos. NIH DK-44319, NIH HO-27691 and NIHDK-48106. The U.S. Government has certain rights to this invention.

1. FIELD OF THE INVENTION

[0003] The present invention relates in general to methods and productsfor initiating an immune response against an antigen, and in particularrelates to transepithelial delivery of antigens to provoke tolerance andimmunity.

[0004] The present invention further relates to methods and products forthe transepithelial delivery of therapeutics. In particular, theinvention relates to methods and compositions for the delivery oftherapeutics conjugated to a FcRn binding partner to intestinalepithelium, mucosal epithelium and epithelium of the lung. The presentinvention further relates to the synthesis, preparation and use of theFcRn binding partner conjugates as, or in, pharmaceutical compositionsfor oral systemic delivery of drugs and vaccines.

2. BACKGROUND OF THE INVENTION

[0005] The immune system of a mammal develops during gestation andbecomes active in the late mammalian fetus. Although active, it stillmight be characterized as ‘immature’ because it has not been challengedto any significant extent by antigens; the fetus is largely protectedfrom antigens by the mother. This ‘immature’ immune system, however, issupplemented by the transfer of maternal immunoglobulin to the fetus (orin some cases to the neonate) to provide humoral immunity during thefirst weeks of independent life.

[0006] Rats and mice receive most maternal immunoglobulin G (IgG) bysuckling from colostrum and milk, although some is acquired prenatally.Cattle also receive IgG from colostrum. In rabbits, IgG is transportedto the fetus across the yolk sac. Little is know about the transfer ofIgG to the fetus or neonate in humans. Most evidence suggests that humanmothers transfer humoral immunity to an offspring only before birth,although IgA transferred to a neonate via breast milk is believed toplay a role in protecting the neonate against enteric infection.

[0007] The delivery of maternal IgG to the mammalian fetus and/orneonate requires transport across an epithelial barrier which is largelyimpervious to macromolecules. The transport of macromolecules acrosssuch an epithelial barrier may occur by non-specific and specificreceptor-mediated mechanisms. Receptor non-specific mechanisms arerepresented by paracellular sieving events, the efficiency of which areinversely related to the molecular weight of the transported molecule.Transport of macromolecules such as IgG across this paracellular pathwayis highly inefficient. Descriptions of receptor-mediated transport ofimmunoglobulins through intestinal epithelial cells are limited thus farto the polymeric immunoglobulin receptor and the enterocyte receptor ofIgG (a major histocompatibility complex (MHC) class I related Fcreceptor). These two receptor systems differ in their specificity forimmunoglobulin isotype, in their direction of immunoglobulin transportacross the epithelial cell and in their tissue-specific expression. Bothmay play a role in molding the immature immune system.

[0008] The polymeric immunoglobulin receptor is expressed on thebasolateral surfaces of enterocytes, hepatocytes and/or biliary ductepithelial cells. It transports polymeric IgA and IgM to the apical(luminal) surfaces, concentrating these immunoglobulins forantimicrobial defense and antigen exclusion.

[0009] The enterocyte receptor for IgG, which has homology to the MHCclass I heavy chain and is associated with beta₂-microglobulin (β₂M), isexpressed on neonatal enterocytes of the rat and mouse. IgG istransported transcellularly in a luminal to serosal direction across theintestinal epithelium of these rodent neonates. On the apical surface ofthe enterocyte, the Fc portion of IgG is bound to the enterocytereceptor at the relatively acidic pH of the lumen (about pH 6.0).Following transcytosis to the basolateral plasma membrane, discharge ofthe immunoglobulin occurs at the relatively neutral pH of theinterstitial fluids (about pH 7.4). The rodent neonatal Fc receptor(FcRn) therefore could be responsible for delivery of maternal IgG tothe neonate and as such may be responsible for the passive acquisitionof IgG during this period.

[0010] In humans, maternal IgG is actively transported across theplacenta. The receptor responsible for this transport has been soughtfor many years. Several IgG-binding proteins have been isolated fromplacenta. FcγRII was detected in placental endothelium and FcγRIII insyncytiotrophoblasts. Both of these receptors, however, showed arelatively low affinity for monomeric IgG. Recently, the isolation fromplacenta of a cDNA encoding a human homolog of the rat and mouseenterocyte receptor for IgG was reported. (Story, C. M. et al., J. Exp.Med., Vol. 180:2377-2381, December 1994) The complete nucleotide anddeduced amino acid sequence is reported. This Fc receptor for IgG may beresponsible for the transport of maternal IgG to the human fetus (andeven possibly to the neonate), as the molecule is highly homologous overits open reading frame with the rat FcRn sequence (69% nucleotideidentity and 65% predicted amino acid identity). So called passiveimmunization in the human fetus (and possibly in the human neonate) nowmay become better understood.

[0011] In contrast to passive immunization which involves supplementinga host's immune system with antibodies derived from another, activeimmunization involves stimulation of the host's own immune system togenerate in vivo the desired immune response. The most widely practicedmethods of active immunization in children and adults involve injectionsof an immunogen, once as an initial dose and then at least once again asa booster dose. These methods suffer many serious drawbacks, includingthe risks associated with the use of needles that can transmit diseasessuch as AIDS and hepatitis. (When tolerizing a patient against anallergen, the problems are compounded in that repeated injections over along period of time often are required.) These methods also do notnecessarily trigger adequately the first line of defense against manypathogens, that is, mucosal immunity. Mucous membranes line the airways,the reproductive system and the gastrointestinal tract, and this mucosalsurface represents the first portal of entry for many diseases. An oralvaccine that is easy to deliver and that triggers mucosal immunity wouldbe highly desirable.

[0012] Immunization using oral vaccines is problematic. Often little orno immune response is achieved. To enhance the immune response, antigensof interest have been coupled to carriers that are known to be stronglyimmunogenic. For example, researchers have delivered antigens usingBacille Calmette-Gurein (BCG) as a carrier; BCG is a bacteriumoriginally used as an oral vaccine against tuberculosis. A problem withsuch carriers is that the patient will develop antibodies against thecarrier itself, which can be troublesome if the carrier is used againfor delivering a different antigen to the same patient. To date, nogeneral strategy for oral vaccine: has proven successful.

[0013] Immunoglobulin and portions thereof in the past have beenconjugated to drugs and imaging agents to target and destroy cellpopulations and to extend the half-lives of certain agents. Immunotoxinsare an example of such conjugates. Such conjugates, however, have neverbeen proposed as useful for initiating an immune response.

[0014] A small body of work has focused on the tolerogenic capacity ofimmunoglobulins coupled to oligonucleotides or proteins characteristicof autoimmune diseases. (See PCT WO 91/08773). This work is based uponthe notion that the induction of tolerance may be strongly influenced bycarrier moieties and that immunoglobulin carriers appear to be stronglytolerogenic. Isologous IgG is the preferred carrier, and intravenousadministration was the mode used for delivering the conjugates of IgG.Although this body of work extends for more than a decade, oraladministration is mentioned only once and only for conjugates where IgAis the immunoglobulin carrier. Thus, although tolerogenic immunoglobulinconjugates are known in the art, such conjugates have never beensuggested as agents for inducing a robust response against an antigencharacteristic of a pathogen. (To the contrary, the art suggests thatsuch conjugates, if anything, would tolerize a subject against apathogen which would be highly undesirable). In addition, it never hasbeen suggested that such conjugates would be effective tolerogens whenthe immunoglobulin is IgG and the mode of delivery is oral delivery.

3. SUMMARY OF THE INVENTION

[0015] The invention involves the discovery that antigens may be coupledto molecules that bind to the FcRn receptor, such as immunoglobulins, orportions thereof, and delivered across epithelial barriers by activetransport through the enterocyte via FcRn receptors. The immunoglobulinor portion thereof binds to the FcRn receptor and acts as a carrier forthe antigen as the immunoglobulin or portion thereof is transportedacross the epithelial barrier by FcRn mediated-transport. The FcRnreceptor is present in the human epithelial tissue of children andadults, and the invention therefore permits effective strategies forimmunizing humans.

[0016] According to one aspect of the invention, a method for modulatingthe immune system of a mammal is provided. An effective amount of aconjugate of an antigen and a FcRn binding partner is administered to anepithelial barrier of a mammal in need of such immune modulation. Theantigen is selected from the group consisting of: an antigen that ischaracteristic of a pathogen, an antigen that is characteristic of anautoimmune disease, an antigen that is characteristic of an allergen andan antigen that is characteristic of a tumor.

[0017] The FcRn binding partners of the present invention may beutilized for the delivery of a wide variety of compounds andtherapeutics and bioactive substances, including but not limited to,chemotherapy agents for the treatment of cancer, cytokines, includinginterferon; and hormones including insulin, human growth hormone (HGH),fertility drugs, calcitonin, calcitriol and other bioactive steroids.The FcRn binding partners of the present invention may further beutilized for the targeted delivery of a delivery vehicle, such asliposomes.

[0018] In preferred embodiments, the FcRn binding partner isnon-specific IgG or a FcRn binding fragment of IgG. Most preferably theFcRn binding partner is an Fc fragment of IgG. It also is preferred thatthe antigen be covalently coupled to the FcRn binding partner.Preferably the conjugate is administered orally to the intestinalepithelium, in an aerosol to the lungs or intranasally. Suchpreparations may be nonaseptic. Supplementary potentiating agents, asdescribed below, may be administered in addition.

[0019] In a preferred embodiment, when the compound to be delivered is apeptide or protein, the FcRn binding partner protein conjugate may besynthesized as a recombinant fusion protein. Examples of peptides andproteins which may be conjugated in this manner include cytokines;chemokines; growth factors, insulin, erythropoietin (EPO),neuropeptides, neuropeptide Y, neurotensin, transforming growth factorα, transforming growth factor β, interferon (IFN), and hormones.

[0020] The pharmaceutical compositions of the present invention relateto FcRn binding partners conjugated to bioactive substances, includingvaccines or drugs for oral, sublingual or intranasal systemic delivery.The pharmaceutical preparation of the present invention includes aconjugate of an antigen and a FcRn binding partner, wherein the antigenis selected from the group consisting of: an antigen that ischaracteristic of a pathogen, an antigen that is characteristic of anautoimmune disease, an antigen that is characteristic of an allergen andan antigen that is characteristic of a tumor. The pharmaceuticalpreparation of the present invention includes a conjugate of a drug ortherapeutic and a FcRn binding partner. The preferred-FcRn bindingpartners are as described above. The conjugate is present in an amounteffective for modulating the immune response of a mammal. Thepharmaceutical preparation of the present invention also includes apharmaceutically acceptable carrier. When the antigen is characteristicof an autoimmune disease or an allergen, then the pharmaceuticalpreparations of the invention must be formulated in unit dosage formconstructed and arranged for delivery to an epithelial carrier such asfor oral delivery to the intestinal epithelium, aerosol delivery to thepulmonary epithelium and intranasal delivery to the nasal epithelium.Thus tablets containing IgG (or an FcRn binding portion thereof) coupledto any of the antigens as characterized above are embraced by thepresent invention.

[0021] The foregoing pharmaceutical preparations may be deliveredtogether with supplementary potentiating agents including adjuvants,cytokines, bioadhesives and the like. The supplementary potentiatingagents themselves may be coupled to a FcRn binding partner to facilitatethe delivery of such agents across the epithelial barrier. Preferredmodes of administration in general include oral dosages to theintestinal epithelium, aerosols to the lungs and intranasal dosages.

[0022] The present invention further relates to the synthesis,preparation and use of the FcRn binding partner conjugates of thepresent invention as, or in, pharmaceutical compositions for oral andintranasal systemic delivery of drugs and vaccines. The synthesis of theFcRn binding partner conjugates of the present invention comprisescovalently coupling an antigen or a supplementary potentiating agent toan FcRn binding partner, wherein the antigen or supplementarypotentiating agent is selected as described above. The synthesis of theFcRn binding partner conjugates of the present invention alternativelycomprises covalently coupling a FcRn binding partner to a therapeutic ordrug. Further, the synthesis of the FcRn binding partner conjugates ofthe present invention comprise covalently coupling a FcRn bindingpartner to a delivery vehicle, e.g. liposomes. The preferred FcRnbinding partner also is as described above. The conjugates then can beused to prepare the pharmaceutical preparations of the presentinvention.

[0023] In yet another aspect of the invention, the conjugate includingthe antigen crosses the epithelial barrier in an amount at least doublethe extent that the antigen crosses the epithelial barrier in anunconjugated form. It thus is an object of the invention to develop amechanism for increasing the ability of an antigen to cross anepithelial barrier.

[0024] Another object of the invention is to develop a new class oforally active immunogens and toleragens.

[0025] Another object of the invention is to develop improved methodsfor stimulating mucosal immunity.

[0026] These and other aspects of the invention are described in greaterdetail below.

4. BRIEF DESCRIPTION OF DRAWING

[0027]FIG. 1. Nucleotide and Amino Acid Sequence of Fc fragment of humanIgG.

5. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention relates to FcRn binding partners modifiedfor the targeted delivery of vaccines, antigens, drugs, therapeutics andliposomes to epithelial barriers. The invention involves the discoverythat the human FcRn receptor is active in adult epithelial tissue andthe discovery that FcRn binding partners such as IgG or Fc fragments canbe used to transport other molecules, including antigens, acrossepithelial barriers. In this manner, “FcRn binding partners”, such asIgG or an FcRn binding portion thereof can be used to deliver an antigenor a therapeutic across an epithelial systemic circulation therebyeliciting a beneficial response or effect, e.g., an immune response.

[0029] The invention is useful whenever it is desirable to enhance thedelivery of an antigen across an epithelial barrier to the immunesystem. The invention thus may be used to deliver antigens acrossintestinal epithelial tissue, lung epithelial tissue and other mucosalsurfaces including nasal surfaces, vaginal surfaces, colon surfaces andbinary tree surfaces. The invention may be used to modulate a subject'simmune system such as by stimulating a humoral antibody response againstan antigen, by stimulating T cell activity, or by stimulating toleranceto an antigen. As used herein, subject means: humans, primates, horses,cows, sheep, pigs goats, dogs, cats, chickens and rodents. Whendelivering, tumor antigens, the invention may be used to treat subjectshaving disease amenable to immunity mediated rejection, such asnon-solid tumors or solid tumors of small size. It is also contemplatedthat delivery of tumor antigens by the methods described herein will beuseful for treatment subsequent to removal of large solid tumors. Theinvention may also be used to treat subjects who are suspected of havingcancer.

[0030] The invention is also useful whenever it is desirable to achievesystemic delivery of a therapeutic or drug or delivery vehicle orprotein or combinations thereof, across an epithelial barrier tosystemic circulation. The invention, thus may be used to delivertherapeutics across intestinal epithelial tissue, lung epithelialtissue, and other mucosal epithelial surfaces including nasal surfaces,vaginal surfaces, colon and rectal surfaces and binary free surfaces.The invention may be used to administer a therapeutic to elicit abeneficial effect. The FcRn binding partner conjugates are designed todeliver a wide variety of therapeutics including RNA and DNA nucleotidesas used, for example, in gene therapy, peptides, carbohydrates and smallmolecules. These therapeutics include but are not limited to, anticancerand chemotherapeutic drugs, e.g., doxorubicin; anti-inflammatory drugs,e.g., steroids; drugs for the treatment of cardiovascular disease, e.g.,cholinesterase inhibitors; drugs for the treatment of disorders relatedto viral infection, e.g. hepatic cirrhosis resulting from hepatitisinfection; drugs for the treatment of weight disorders, e.g.,amphetamines; antibacterial agents, antifungal agents, cytokines,fertility drugs, antibiotics, hormones, steroids, etc. The inventioninvolves the formation of a conjugate of an FcRn binding partner and anantigen or a therapeutic or drug. By conjugate it is meant two or moreentities bound to one another by any physiochemical means, including,but not limited to, hydrophobic interaction, covalent interaction,hydrogen bond interaction, or ionic interaction between a bioactivesubstance, such as, an antigen or a therapeutic and the non-specifichydrophobic portions of an antibody molecule, antibody-antigen specificbinding and covalent coupling. The nature of the preferred bonding willdepend, among other things, upon the mode of administration and thepharmaceutical carriers used to deliver the conjugate to the selectedepithelial barrier. For example, some bonds are not as well suited asothers to withstand certain environments such as the stomach, but can beprotected therefrom by delivery systems which bypass the stomach. It, ofcourse, is important that the bond between the FcRn binding partner andthe antigen be of such a nature that it does not destroy the ability ofthe FcRn binding partner to bind to the FcRn receptor. Such bonds arewell known to those of ordinary skill in the art examples are providedin greater detail below. The conjugate further may be formed as a fusionprotein, also discussed in greater detail below.

[0031] 5.1. FcRn Binding Partners

[0032] An FcRn binding partner means any entity that can be specificallybound by the FcRn receptor with consequent active transport by the FcRnreceptor of the FcRn binding partner. As mentioned above, the FcRnreceptor has been isolated for several mammalian species, includinghumans. The sequence of the human FcRn, rat FcRn and mouse FcRn may befound in Story, C. M. et al, J. Exp. Med., vol. 180:2377-2381, December1994. The FcRn receptor molecule now is well

[0033] characterized. The FcRn receptor binds IgG (but not otherimmunoglobulin classes such as IgA, IgD, IgM and IgE) at a relativelylower pH, actively transports the IgG transcellularly in a luminal toserosal direction, and then releases the IgG at a relatively higher pHfound in the interstitial fluids. As will be recognized by those ofordinary skill in the art, FcRn receptors can be isolated by cloning orby affinity purification using, for example, monoclonal antibodies. Suchisolated FcRn receptors then can be used to identify and isolate FcRnbinding partners, as described below.

[0034] FcRn binding partners of the present invention encompass anyentity that can be specifically bound by the FcRn receptor, includingwhole IgG, the Fc fragment of IgG and other fragments of IgG thatinclude the complete binding region for the FcRn receptor. The region ofthe Fc portion of IgG that binds to the FcRn receptor has been describedbased upon X-ray crystallography (Burmaister, W. P. et al., Nature,1994; 372:379-378.) The major contact area of Fc with the FcRn receptoris near the junction of the C_(H)2 and C_(H)3 domains. Potentialcontacts are residues 248, 250-257, 272, 285, 288, 290-291, 308-311 and314 in C_(H)2 and 385-387, 428 and 433-436 in C_(H)3. (These sites aredistinct from those identified by subclass comparison or bysite-directed mutagenesis as important for Fc binding to leukocyte FcγRIand FcγRII.) The foregoing Fc—FcRn contacts are all within a single Igheavy chain. It has been noted previously that two FcRn receptors canbind a single Fc molecule. The crystallographic data suggest that insuch a complex, each FcRn molecule binds a single polypeptide of the Fchomodimer.

[0035] In an embodiment of the present invention, FcRn binding partnersother than whole IgG may be used to transport therapeutics across theepithelial barrier. In such an embodiment, it is preferred that a FcRnbinding partner is chosen which binds the FcRn with higher affinity thanwhole IgG. Such a FcRn binding partner has utility in utilizing the FcRnto achieve active transport of a conjugated therapeutic across theepithelial barrier, and also has utility to prevent the binding andtransport of whole IgG across the epithelial barrier via the FcRn. TheFcRn binding activity of these higher affinity FcRn binding partners maybe measured using standard assays known to those skilled in the art,including: (a) transport assays using polarized cells that naturallyexpress the FcRn, or have been genetically engineered to express theFcRn or the alpha chain of the FcRn; (b) protein:protein binding assaysusing soluble FcRn or fragments thereof, or immobilized FcRn; (c)binding assays utilizing polarized or non-polarized cells that naturallyexpress the FcRn, or have been genetically engineered to express theFcRn or the alpha chain of the FcRn.

[0036] 5.1.1. Recombinant Synthesis of FcRn Binding Partners

[0037] In accordance with the present invention, the FcRn bindingpartner may be produced by recombinant genetic engineering techniques.Within the scope of the invention are nucleotide sequences encodinghuman FcRn binding partners. The FcRn binding partners include wholeIgG, the Fc fragment of IgG and other fragments of IgG that include thecomplete binding region for the FcRn. The major contact sites includeamino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311 and314 of the C_(H)2 and amino acid residues 385-387, 428 and 433-436 ofthe C_(H)3. Therefore in a preferred embodiment of the present inventionare nucleotide sequences encoding regions of the IgG Fc spanning theseamino acid residues.

[0038] Given the foregoing information, those of ordinary skill in theart will readily recognize that the Fc region of IgG can be modifiedaccording to well-recognized procedures such as site-directedmutagenesis and the like to yield modified IgG or modified Fc fragmentsor portions thereof that will be bound by the FcRn receptor. Suchmodifications include modifications remote from the FcRn contact sitesas well as modifications within the contact sites that preserve or evenenhance binding. In addition, other binding partners can be identifiedand isolated. Antibodies or portions thereof specific for the FcRnreceptor and capable of being transported by FcRn once bound can beidentified and isolated using well established techniques. Likewise,randomly generated molecularly diverse libraries can be screened andmolecules that are bound and transported by FcRn receptors can beisolated using conventional techniques. It is not intended that theinvention be limited by the selection of any particular FcRn bindingpartner.

[0039] If the peptide is composed entirely of gene-encoded amino acids,or a portion of it is so composed, the peptide or the relevant portionmay also be synthesized using conventional recombinant geneticengineering techniques.

[0040] For recombinant production, a polynucleotide sequence encodingthe FcRn binding partner is inserted into an appropriate expressionvehicle, i.e., a vector which contains the necessary elements for thetranscription and translation of the inserted coding sequence, or in thecase of an RNA viral vector, the necessary elements for replication andtranslation. The expression vehicle is then transfected into a suitabletarget cell which will express the peptide. Depending on the expressionsystem used, the expressed peptide is then isolated by procedureswell-established in the art. Methods for recombinant protein and peptideproduction are well known in the art (see, e.g., Maniatis et al., 1989,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley Interscience, N.Y.).

[0041] To increase efficiency of production, the polynucleotide can bedesigned to encode multiple units of the FcRn binding partner separatedby enzymatic cleavage sites. The resulting polypeptide can be cleaved(e.g., by treatment with the appropriate enzyme) in order to recover thepeptide units. This can increase the yield of peptides driven by asingle promoter. When used in appropriate viral expression systems, thetranslation of each peptide encoded by the mRNA is directed internallyin the transcript; e.g., by an internal ribosome entry site, IRES. Thus,the polycistronic construct directs the transcription of a single, largepolycistronic mRNA which, in turn, directs the translation of multiple,individual peptides. This approach eliminates the production andenzymatic processing of polyproteins and may significantly increaseyield of peptide driven by a single promoter.

[0042] A variety of host-expression vector systems may be utilized toexpress the FcRn binding partners described herein. These include, butare not limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage DNA or plasmid DNA expression vectorscontaining an appropriate coding sequence; yeast or filamentous fungitransformed with recombinant yeast or fungi expression vectorscontaining an appropriate coding sequence; insect cell systems infectedwith recombinant virus expression vectors (e.g., baculovirus) containingan appropriate coding sequence; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus ortobacco mosaic virus) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing an appropriate coding sequence; oranimal cell systems.

[0043] The expression elements of the expression systems vary in theirstrength and specificities. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationelements, including constitutive and inducible promoters, may be used inthe expression vector. For example, when cloning in bacterial systems,inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac(ptrp-lac hybrid promoter) and the like may be used; when cloning ininsect cell systems, promoters such as the baculovirus polyhedronpromoter may be used; when cloning in plant cell systems, promotersderived from the genome of plant cells (e.g., heat shock promoters; thepromoter for the small subunit of RUBISCO; the promoter for thechlorophyll a/b binding protein) or from plant viruses (e.g., the 35SRNA promoter of CaMV; the coat protein promoter of TMV) may be used;when cloning in mammalian cell systems, promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5 K promoter) may be used; when generating cell lines thatcontain multiple copies of expression product, SV40-, BPV- and EBV-basedvectors may be used with an appropriate selectable marker.

[0044] In cases where plant expression vectors are used, the expressionof sequences encoding linear or non-cyclized forms of the cyclicpeptides of the invention may be driven by any of a number of promoters.For example, viral promoters such as the 35S RNA and 19S RNA promotersof CaMV (Brisson et al., 1984, Nature 310:511-514), or the coat proteinpromoter of TMV (Takamatsu et al., 1987, EMBO J. 6:307-311) may be used;alternatively, plant promoters such as the small subunit of RUBISCO(Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al., 1984,Science 224:838-843) or heat shock promoters, e.g., soybean hspl7.5-E orhspl7.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565) may be used.These constructs can be introduced into plant cells using Ti plasmids,Ri plasmids, plant virus vectors, direct DNA transformation,microinjection, electroporation, etc. For reviews of such techniquessee, e.g., Weissbach & Weissbach, 1988, Methods for Plant MolecularBiology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson &Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.

[0045] In one insect expression system that may be used to produce thepeptides of the invention, Autographa californica nuclear polyhidrosisvirus (AcNPV) is used as a vector to express the foreign genes. Thevirus grows in Spodoptera frugiperda cells. A coding sequence may becloned into non-essential regions (for example the polyhedron gene) ofthe virus and placed under control of an AcNPV promoter (for example,the polyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (e.g., see Smith et al., 1983, J. Virol. 46:584;Smith, U.S. Pat. No. 4,215,051). Further examples of this expressionsystem may be found in Current Protocols in Molecular Biology, Vol. 2,Ausubel et al., eds., Greene Publish. Assoc. & Wiley Interscience.

[0046] In mammalian host cells, a number of viral based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. (e.g., See Logan & Shenk, 1984, Proc. Natl.Acad. Sci. (USA) 81:3655-3659). Alternatively, the vaccinia 7.5 Kpromoter may be used, (see, e.g., Mackett et al., 1982, Proc. Natl.Acad. Sci. (USA) 79:7415-7419; Mackett et al., 1984, J. Virol.49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931).

[0047] 5.2. FcRn Binding Partners Conjugated to Antigens for VaccineDelivery

[0048] The FcRn binding partner is conjugated with an antigen. Anantigen as used herein falls into four classes: 1) antigens that arecharacteristic of a pathogen; 2) antigens that are characteristic of anautoimmune disease; 3) antigens that are characteristic of an allergen;and 4) antigens that are characteristic of a tumor. Antigens in generalinclude polysaccharides, glycolipids, glycoproteins, peptides, proteins,carbohydrates and lipids from cell surfaces, cytoplasm, nuclei,mitochondria and the like.

[0049] Antigens that are characteristic of pathogens include antigensderived from viruses, bacteria, parasites or fungi. Examples ofimportant pathogens include vibrio choleras, enterotoxigenic Escherichiacoli, rotavirus, Clostridium difficile, Shigella species, Salmonellatyphi, parainfluenza virus, influenza virus, Streptococcus pneumonias,Borella burgdorferi, HIV, Streptococcus mutans, Plasmodium falciparum,Staphylococcus aureus, rabies virus and Epstein-Barr virus.

[0050] Viruses in general include but are not limited to those in thefollowing families: picornaviridae; caliciviridae; togaviridae;flaviviridae; coronaviridae; rhabdoviridae; filoviridae;paramyxoviridae; orthomyxoviridae; bunyaviridae; arenaviridae;reoviridae; retroviridae; hepadnaviridae; parvoviridae; papovaviridae;adenoviridae; herpesviridae; and poxyviridae.

[0051] Bacteria in general include but are not limited to: P.aeruginosa; E. coli, Klebsiella sp.; Serratia sp.; Pseudomanas sp.; P.cepacia; Acinetobacter sp.; S. epidermis; E. faecalis; S. pneumonias; S.aureus; Haemophilus sp.; Neisseria Sp.; N. meningitidis; Bacteroidessp.; Citrobacter sp.; Branhamella sp.; Salmonelia sp.; Shigella sp.; S.pyogenes; Proteus sp.; Clostridium sp.; Erysipelothrix sp.; Lesteriasp.; Pasteurella multocida; Streptobacillus sp.; Spirillum sp.;Fusospirocheta sp.; Treponema pallidum; Borrelia sp.; Actinomycetes;Mycoplasma sp.; Chlamydia sp.; Rickettsia sp.; Spirochaeta; Legionellasp.; Mycobacteria sp.; Ureaplasma sp.; Streptomyces sp.; Trichomorassp.; and P. mirabilis.

[0052] Parasites include but are not limited to: Plasmodium falciparum,P. vivax, P. ovale, P. malaria; Toxoplasma gondii; Leishmania mexicana,L. tropica, L. major, L. aethiopica, L. donovani, Trypanosoma cruzi, T.brucei, Schistosoma mansoni, S. haematobium, S. japonium; Trichinellaspiralis; Wuchereria bancrofti; Brugia malayli; Entamoeba histolytica;Enterobius vermiculoarus; Taenia solium, T. saginata, Trichomonasvaginatis, T. hominis, T. tenax; Giardia lamblia; Cryptosporidiumparvum; Pneumocytis carinii, Babesia bovis, B. divergens, B. microti,Isospore belli, L hominis; Dientamoeba fragiles; Onchocerca volvulus;Ascaris lumbricoides; Necator americanis; Ancylostoma duodenale;Strongyloides stercoralis; Capillaria philippinensis; Angiostrongyluscantonensis; Hymenolepis nana; Diphyllobothrium latum; Echinococcusgranulosus, E. multilocularis; Paragonimus westermani, P. caliensis;Chlonorchis sinensis; Opisthorchis felineas, G. Viverini, Fasciolahepatica Sarcoptes scabiei, Pediculus humanus; Phthirius pubis; andDermatobia hominis.

[0053] Fungi in general include but are not limited to: Cryptococcusneoformans; Blastomyces dermatitidis; Aiellomyces dermatitidis;Histoplasfria capsulatum; Coccidioides immitis; Candids species,including C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondiiand C. krusei, Aspergillus species, including A. fumigatus, A. flavusand A. niger, Rhizopus species; Rhizomucor species; Cunninghammellaspecies; Apophysomyces species, including A. saksenaea, A. mucor and A.absidia; Sporothrix schenckii, Paracoccidioides brasiliensis;Pseudallescheria boydii, Torulopsis glabrata; and Dermatophyres species.

[0054] Antigens that are characteristic of autoimmune disease typicallywill be derived from the cell surface, cytoplasm, nucleus, mitochondriaand the like of mammalian tissues. Examples include antigenscharacteristic of uveitis (e.g. S antigen), diabetes mellitus, multiplesclerosis, systemic lupus erythematosus, Hashimoto's thyroiditis,myasthenia gravis, primary myxoedema, thyrotoxicosis, rheumatoidarthritis, pernicious anemia, Addison's disease, scleroderma, autoimmuneatrophic gastritis, premature menopause (few cases), male infertility(few cases), juvenile diabetes, Goodpasture's syndrome, pemphigusvulgaris, pemphigoid, sympathetic opthalmia, phacogenic uveitis,autoimmune haemolytic anemia, idiopathic thrombocylopenic purpura,idiopathic feucopenia, primary biliary cirrhosis (few cases), ulcerativecolitis, Siogren's syndrome, Wegener's granulomatosis,poly/dermatomyositis, and discold lupus erythromatosus.

[0055] Antigens that are allergens are generally proteins orglycoproteins, although allergens may also be low molecular weightallergenic haptens that induce allergy after covalently combining with aprotein carrier (Remington's Pharmaceutical Sciences). Allergens includeantigens derived from pollens, dust, molds, spores, dander, insects andfoods. Specific examples include the urushiols (pentadecylcatechol orheptadecyicatechol) of Toxicodendron species such as poison ivy, poisonoak and poison sumac, and the sesquiterpenoid lactones of ragweed andrelated plants.

[0056] Antigens that are characteristic of tumor antigens typically willbe derived from the cell surface, cytoplasm, nucleus, organelles and thelike of cells of tumor tissue. Examples include antigens characteristicof tumor proteins, including proteins encoded by mutated oncogenes;viral proteins associated with tumors; and tumor mucins and glycolipids.Tumors include, but are not limited to, those from the following sitesof cancer and types of cancer: lip, nasopharynx, pharynx and oralcavity, esophagus, stomach, colon, rectum, liver, gall bladder, binarytree, pancreas, larynx, lung and bronchus, melanoma of skin, breast,cervix, uteri, uterus, ovary, bladder, kidney, brain and other parts ofthe nervous system, thyroid, prostate, testes, Hodgkin's disease,non-Hodgkin's lymphoma, multiple myeloma and leukemia. Viral proteinsassociated with tumors would be those from the classes of viruses notedabove. Antigens characteristic of tumors may be proteins not usuallyexpressed by a tumor precursor cell, or may be a protein which isnormally expressed in a tumor precursor cell, but having a mutationcharacteristic of a tumor. An antigen characteristic of a tumor may be amutant variant of the normal protein-having an altered activity orsubcellular distribution. Mutations of genes giving rise to tumorantigens, in addition to those specified above, may be in the codingregion, 5′ or 3′ noncoding regions, or introns of a gene, and may be theresult of point mutations frameshifts, deletions, additions,duplications, chromosomal rearrangements and the like. One of ordinaryskill in the art is familiar with the broad variety of alterations tonormal gene structure and expression which gives rise to tumor antigens.Specific examples of tumor antigens include: proteins such asIg-idiotype of B cell lymphoma, mutant cyclin-dependent kinase 4 ofmelanoma, Pmel-17 (gp 100) of melanoma, MART-1 (Melan-A) of melanoma,p15 protein of melanoma, tyrosinase of melanoma, MAGE 1, 2 and 3 ofmelanoma, thyroid medullary, small cell lung cancer, colon and/orbronchial squamous cell cancer, BAGE of bladder, melanoma, breast, andsquamous-cell carcinoma, gp75 of melanoma, oncofetal antigen ofmelanoma; carbohydrate/lipids such as muci mucin of breast, pancreas,and ovarian cancer, GM2 and GD2 gangliosides of melanoma; oncogenes suchas mutant p53 of carcinoma, mutant ras of colon cancer and HER21neuproto-onco-gene of breast carcinoma; viral products such as humanpapilloma virus proteins of squamous cell cancers of cervix andesophagus. It is also contemplated that proteinaceous tumor antigens maybe presented by HLA molecules as specific peptides derived from thewhole protein. Metabolic processing of proteins to yield antigenicpeptides is well known in the art; for example see U.S. Pat. No.5,342,774 (Boon et al.). The present method thus encompasses delivery ofantigenic peptides and such peptides in a larger polypeptide or wholeprotein which give rise to antigenic peptides. Delivery of antigenicpeptides or proteins may give rise to humoral or cellular immunity.

[0057] Generally, subjects can receive an effective amount of the tumorantigen, and/or peptide derived therefrom by one or more of the methodsdetailed below. Initial doses can be followed by booster doses,following immunization protocols standard in the art. Delivery of tumorantigens thus may stimulate proliferation of cytolytic T lymphocytes.

[0058] In the cases of protein and peptide antigens, covalent linking toan FcRn partner is intended to include linkage by peptide bonds in asingle polypeptide chain. Established methods (Sambrook et al.,Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, ColdSpring Harbor, N.Y. 1989) would be used to engineer DNA encoding afusion protein comprised of the antigenic peptide or protein and an FcRnpartner. This DNA would be placed in an expression vector and introducedinto bacterial or eukaryotic cells by established methods. The fusionprotein would be purified from the cells or from the culture medium byestablished methods.

[0059] In accordance with the present invention, a conjugate may beformed as a fusion protein. Conjugates include fusions of the FcRnbinding partner to a protein, peptide or protein derivative such asthose listed herein including, but not limited to, antigens, allergens,pathogens or to other proteins or protein derivatives of potentialtherapeutic interest such as growth factors, colony stimulating factors,growth inhibitory factors, signaling molecules, hormones, steroids,neurotransmitters, or morphogens that would be of use when deliveredacross an epithelial barrier.

[0060] By way of example, but not limitation, proteins used in fusionproteins to synthesize conjugates may include Platelet Derived GrowthFactor (PDGF) (U.S. Pat. No. 4,766,073), Platelet Derived EndothelialCell Growth Factor (PD-ECGF) (U.S. Pat. No. 5,227,302), Human pituitaryGrowth Hormone (HGH) (U.S. Pat. No. 3,853,833), Transforming GrowthFactor Beta (TGFβ) (U.S. Pat. No. 5,168,051), Transforming Growth FactorAlpha (TGFα) (U.S. Pat. No. 5,633,147), Keratinocyte Growth Factor (KGF)(U.S. Pat. No. 5,731,170), Insulin-like Growth Factor I (IGF-I) (U.S.Pat. No. 4,963,665), Epidermal Growth Factor (EGF) (U.S. Pat. No.5,096,825), Erythropoietin (EPO) (U.S. Pat. No. 4,703,008),Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) (U.S. Pat. No.5,200,327), M-CSF (U.S. Pat. No. 5,171,675), Colony Stimulating Factor-1(CSF-1) (U.S. Pat. No. 4,847,201), Steel factor, Calcitonin, AP-1proteins (U.S. Pat. No. 5,238,839), Brain Derived Neurotrophic Factor(BDNF) (U.S. Pat. No. 5,229,500). All of the references cited above areincorporated herein by reference in their entirety.

[0061] In one embodiment, fusion proteins of the present invention areconstructed in which the conjugate consists of an Fc fragment (startingwith the amino acids D-K-T-H at the N-terminus of the CH1 region,including the hinge and CH2 regions, and continuing through the S-P-G-Ksequence in CH3 region) fused to one of the above listed proteins. As anexample, the nucleotide sequences encoding Erythropoietin (EPO) arefused to the Fc binding partner.

[0062] In order to engineer the DNA vector encoding the fusion proteinFcRn binding partner conjugate, the mammalian expression vectorscFvE-sec (described in Persic, et.al. GENE 187:1-8 1997 andincorporated herein by reference in its entirety) was linearized withBssH2 and Not1 and the linearized plasmid was gel purified by protocolsstandard in the art.

[0063] The DNA encoding the protein to be fused to the Fc was preparedas follows. The entire (mouse) EPO sequence was removed from theexpression vector (described in Bill et al., Biochem. Biophy. Acta1261:35-43, 1995, incorporated herein by reference in its entirety) bythe restriction enzymes BamH1 and Sal1 sites outside the EPO insert. Thelinearized fragment was gel purified and modified by standard PCR(Polymerase Chain Reaction) protocols and a BssH2 site added adjacent tothe DNA encoding the first N-terminal alanine of mouse EPO (5′ end); theDNA encoding the C-terminal end of mouse EPO was modified with asequence corresponding to an ala-ala-ala linker followed by a Sal1restriction site (3′ end). The EPO fragment was then cut with BssH1 andSal1 and gel purified by standard protocols in the art. The Fc fragmentwas cut with restriction enzymes of SalI and NotI and gel purified. Thethree linearized and gel purified DNAs described above, including thescfve-sec vector, Fc fragment and modified EPO DNAs were ligated into asingle circular vector by a triple ligation using standard protocols inthe art.

[0064] It is noted that the DNA encoding EPO may be replaced with DNAencoding any of the above proteins or other proteins of interest orprotein fragments thereof by standard techniques to create an Fc fusionprotein leading to a conjugate. Similarly this invention allows for thereplacement of DNA encoding the Fc fragment in the vector with DNAencoding a FcRn binding partner as described herein. Additionally, thisinvention allows for the replacement of both the DNA encoding EPO andthe DNA encoding the Fc fragment such that the final vector results in aconjugate that associates with the FcRn.

[0065] In order to demonstrate that a conjugate made by the fusion of aFcRn binding partner and a protein of interest is capable of retainingbiological activity, the example protein above was expressed and assayedfor biological activity of erythropoietin in the following manner. Themammalian expression vector containing the EPO-Fc fusion was transfectedinto CHO (Chinese Hamster Ovary) cells and expressed by standardprotocols in the art. Supernatants of transfected or non-transfected CHOcells were collected and injected subcutaneously into Balbc mice.Reticulocyte counts of mice were obtained by coulter FACs analysis bytechniques known in the field of the art. Results demonstrated that miceinjected with the supernatants of the transfected cells had reticulocyte(mature erythrocytes) counts several fold higher than mice injected withcontrol (untransfected supernatants). Since EPO has been documented tostimulate the production of erythrocytes, the results disclosed hereinsupport the ability of the invention to synthesize biologically activeFcRn binding partner conjugates.

[0066] Similarly, fusion proteins substituting the Fc fragment for analternate FcRn binding partner domain in the vector described abovewould be expected to retain biological activity.

[0067] 5.3. FcRn Binding Partners Conjugated to Therapeutics forSystemic Drug Delivery

[0068] The FcRn binding partners may be conjugated to a variety oftherapeutics or drugs for targeted systemic delivery. The presentinvention encompasses the targeted systemic delivery of biologicallyactive substances.

[0069] As used herein, the term “biologically active substance” refersto eukaryotic and procaryotic cells, viruses, vectors, proteins,peptides, nucleic acids, polysaccharides and carbohydrates, lipids,glycoproteins, and combinations thereof, and synthetic organic andinorganic drugs exerting a biological effect when administered to ananimal. For ease of reference, the term is also used to includedetectable compounds such as radiopaque compounds including air andbarium, magnetic compounds. The active substance can be soluble orinsoluble in water. Examples of biologically active substances includeanti-angiogenesis factors, antibodies, growth factors hormones, enzymes,and drugs such as steroids, anti-cancer drugs or antibiotics.

[0070] In diagnostic embodiments, the FcRn binding partners may also beconjugated to a pharmaceutically acceptable gamma-emitting moiety,including but not limited to, indium and technetium, magnetic particles,radiopaque materials such as air or barium and fluorescent compounds.

[0071] By way of example, and not be limitation, the following classesof drugs may be conjugated to FcRn binding partners for the purposes ofdelivery to epithelial borders:

[0072] Antineoplastic Compounds. Nitrosoureas, e.g., carmustine,lomustine, semustine, strepzotocin; Methylhydrazines, e.g.,procarbazine, dacarbazine; steroid hormones, e.g., glucocorticoids,estrogens, progestins, androgens, tetrahydrodesoxycaricosterone,cytokines and growth factors; Asparaginase.

[0073] Immunoactive Compounds. Immunosuppressives, e.g., pyrimethamine,trimethopterin, penicillamine, cyclosporine, azathioprine;immunostimulants, e.g., levamisole, diethyl dithiocarbamate,enkephalins, endorphins.

[0074] Antimicrobial Compounds. Antibiotics, e.g., β lactam, penicillin,cephalosporins, carbapenims and monobactams, β-lactamase inhibitors,aminoglycosides, macrolides, tetracyclins, spectinomycin; Antimalarials,Amebicides, Antiprotazoal, Antifungals, e.g., amphotericin β, Antiviral,e.g., acyclovir, idoxuridine, ribavirin, trifluridine, vidarbine,gancyclovir.

[0075] Parasiticides. Antihalmintics, Radiopharmaceutics,gastrointestinal drugs.

[0076] Hematologic Compounds. Immunoglobulins; blood clotting proteins;e.g., antihemophilic factor, factor IX complex; anticoagulants, e.g.,dicumarol, heparin Na; fibrolysin inhibitors, tranexamic acid.

[0077] Cardiovascular Drugs. Peripheral antiadrenergic drugs, centrallyacting antihypertensive drugs, e.g., methyldopa, methyldopa HCl;antihypertensive direct vasodilators, e.g., diazoxide, hydralazine HCl;drugs affecting renin-angiotensin system; peripheral vasodilators,phentolamine; antianginal drugs; cardiac glycosides; inodilators; e.g.,amrinone, milrinone, enoximone, fenoximone, imazodan, sulmazole;antidysrhythmic; calcium entry blockers; drugs affecting blood lipids;ranitidine, bosentan, rezulin.

[0078] Respiratory Drugs. Sypathomimetic drugs: albuterol, bitolterolmesylate, dobutamine HCl, dopamine HCl, ephedrine SO, epinephrine,fenfluramine HCl, isoproterenol HCl, methoxamine HCl, norepinephrinebitartrate, phenylephrine HCl, ritodrine HCl; cholinomimetic drugs,e.g., acetylcholine Cl; anticholinesterases, e.g., edrophonium Cl;cholinesterase reactivators; adrenergic blocking drugs, e.g., acebutololHCl, atenolol, esmolol HCl, labetalol HCl, metoprolol, nadolol,phentolamine mesylate, propanolol HCl; antimuscarinic drugs, e.g.,anisotropine methylbromide, atropine SO4, clinidium Br, glycopyrrolate,ipratropium Br, scopolamine HBr.

[0079] Neuromuscular Blocking Drugs. Depolarizing, e.g., atracuriumbesylate, hexafluorenium Br, metocurine iodide, succinylcholine Cl,tubocurarine Cl, vecuronium Br; centrally acting muscle relaxants, e.g.,baclofen.

[0080] Neurotransmitters and neurotransmitter Agents. Acetylcholine,adenosine, adenosine triphosphate, amino acid neurotransmitters, e.g.,excitatory amino acids, GABA, glycine; biogenic amine neurotransmitters,e.g., dopamine, epinephrine, histamine, norepinephrine, octopamine,serotonin, tyramine; neuropeptides, nitric oxide, K+ channel toxins,

[0081] Antiparkinson Drugs. amaltidine HCl, benztropine mesylate, e.g.,carbidopa.

[0082] Diuretic Drugs. Dichlorphenamide, methazolamide,bendroflumethiazide, polythiazide.

[0083] Uterine, Antimigraine Drugs. Carboprost tromethamine, mesylate,methysergide maleate.

[0084] Hormones. Pituitary hormones, e.g., chorionic gonadotropin,cosyntropin, menotropins, somatotropin, iorticotropin, protirelin,thyrotropin, vasopressin, lypressin; adrenal hormones, e.g.,beclomethasone dipropionate, betamethasone, dexamethasone,triamcinolone; pancreatic hormones, e.g., glucagon, insulin; parathyroidhormone, e.g., dihydrochysterol; thyroid hormones, e.g., calcitoninetidronate disodium, levothyroxine Na, liothyronine Na, liotrix,thyroglobulin, teriparatide acetate; antithyroid drugs; estrogenichormones; progestins and antagonists, hormonal contraceptives,testicular hormones; gastrointestinal hormones: cholecystokinin,enteroglycan, galanin, gastric inhibitory polypeptide, epidermal growthfactor-urogastrone, gastric inhibitory polypeptide, gastrin-releasingpeptide, gastrins, pentagastrin, tetragastrin, motilin, peptide YY,secretin, vasoactive intestinal peptide, sincalide.

[0085] Enzymes. Hyaluronidase, streptokinase, tissue plasminogenactivator, urokinase, PGE-adenosine deaminase.

[0086] Intravenous Anesthetics. Droperidol, etomidate, fetanylcitrate/droperidol, hexobarbital, ketamine HCl, methohexital Na,thiamylal Na, thiopental Na.

[0087] Antiepileptics. Carbamazepine, clonazepam, divalproex Na,ethosuximide, mephenytoin, paramethadione, phenytoin, primidone.

[0088] Peptides and proteins. The FcRn binding partners may beconjugated to peptides or polypeptides, e.g., ankyrins, arrestins,bacterial membrane proteins, clathrin, connexins, dystrophin, endothelinreceptor, spectrin, selectin, cytokines; chemokines; growth factors,insulin, erythropoietin (EPO), tumor necrosis factor (TNF),neuropeptides, neuropeptide Y, neurotensin, transforming growth factorα, transforming growth factor β, interferon (IFN), and hormones, growthinhibitors, e.g., genistein, steroids etc; glycoproteins, e.g., ABCtransporters, platelet glycoproteins, GPIb-IX complex, GPIIb-IIIacomplex, vitronectin, thrombomodulin, CD4, CD55, CD58, CD59, CD44,lymphocye function-associated antigen, intercellular adhesion molecule,vascular cell adhesion molecule, Thy-1, antiporters, CA-15-3 antigen,fibronectins, laminin, myelin-associated glycoprotein, GAP, GAP-43. Inthis embodiment of the present invention, the polypeptide therapeuticsmay be covalently conjugated to the FcRn binding partner or the FcRnbinding partner and therapeutic may be expressed as a fusion proteinusing standard recombinant genetic techniques, see Section 5.1.1 infra.

[0089] Cytokines and Cytokine Receptors. Examples of cytokines andreceptors thereof which may be delivered via a FcRn binding parnter orconjugated to an FcRn binding partner in accordance with the presentinvention, include, but are not limited to: Interleukin-1 (IL-1), IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-1 receptor, IL-2 receptor, IL-3receptor, IL-4 receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor,IL-8 receptor, IL-9 receptor, IL-10 receptor, IL-11 receptor, IL-12receptor, IL-13 receptor, IL-14 receptor, IL-15 receptor, IL-16receptor, IL-17 receptor, IL-18 receptor, lymphokine inhibitory factor,macrophage colony stimulating factor, platelet derived growth factor,stem cell factor, tumor growth factor β, tumor necrosis factor,lymphotoxin, Fas, granulocyte colony stimulating factor, granulocytemacrophage colony stimulating factor, interferon α, interferon β,interferon γ.

[0090] Growth Factors and Protein Hormones. Examples of growth factorsand receptors thereof and protein hormones and receptors thereof whichmay be delivered via a FcRn binding parnter or conjugated to an FcRnbinding partner in accordance with the present invention, include, butare not limited to: erythropoietin, angiogenin, hepatocyte growthfactor, fibroblast growth factor, keratinocyte growth factor, nervegrowth factor, tumor growth factor α, thrombopoietin, thyroidstimulating factor, thyroid releasing hormone, neurotrophin, epidermalgrowth factor, VEGF, ciliary neurotrophic factor, LDL, somatomedin,insulin growth factor, insulin-like growth factor I and II.

[0091] Chemokines. Examples of chemokines and receptors thereof whichmay be delivered via a FcRn binding parnter or conjugated to an FcRnbinding partner in accordance with the present invention, include, butare not limited to: ENA-78, ELC, GRO-α, GRO-β, GRO-γ, HRG, LIF, IP-10,MCP-1, MCP-2, MCP-3, MCP-4, MIP-1α, MIP-1β, MIG, MDC, NT-3, NT-4, SCF,LIF, leptin, RANTES, lymphotactin, eotaxin-1, eotaxin-2, TARC, TECK,WAP-1, WAP-2, GCP-1, GCP-2, α-chemokine receptors: CXCR1, CXCR2, CXCR3,CXCR4, CXCR5, CXCR6, CXCR7 β-chemokine receptors: CCR1, CCR2, CCR3,CCR4, CCR5, CCR6, CCR7.

[0092] Chemotherapeutics. The FcRn binding partners may be conjugated tochemotherapy or anti-tumor agents which are effective against varioustypes of human cancers, including leukemia, lymphomas, carcinomas,sarcomas, myelomas etc., such as, doxorubicin, mitomycin, cisplatin,daunorubicin, bleomycin, actinomycin D, neocarzinostatin.

[0093] Antibodies. The FcRn binding partners of the present inventionmay be conjugated to antibodies including, but not limited to: (a)anti-cluster of differentiation antigen CD-1 through CD-166 and theligands or counter receptors for these molecules; (b) anti-cytokineantibodies, e.g., anti-IL-1 through anti-IL-18 and the receptors forthese molecules; (c) anti-immune receptor antibodies, antibodies againstT cell receptors, major histocompatibility complexes I and II, B cellreceptors, selectin killer inhibitory receptors, killer activatingreceptors, OX-40, MadCAM-1, Gly-CAM1, integrins, cadherens,sialoadherens, Fas, CTLA-4, Fcγ-receptors, Fcα-receptors, Fcε-receptors,Fcμ-receptors, and their ligands; (d) anti-metalloproteinase antibodies,e.g., collagenase, MMP-1 through MMP-8, TIMP-1, TIMP-2; anti-celllysis/proinflammatory molecules, e.g., perforin, complement components,prostanoids, nitron oxide, thromboxanes; and (e) anti-adhesionmolecules, e.g., carcioembryonic antigens, lamins, fibronectins.

[0094] Antiviral Agents. The FcRn binding partners may be conjugated toantiviral agents such as reverse transcriptase inhibitors and nucleosideanalogs, e.g. ddI, ddC, 3TC, ddA, AZT; protease inhibitors, e.g.,Invirase, ABT-538; inhibitors of in RNA processing, e.g., ribavirin.

[0095] Specific examples of known therapeutics which may be deliveredvia a FcRn binding partner include, but are not limited to:

[0096] (a) Capoten, Monopril, Pravachol, Avapro, Plavix, Cefzil,Duricef/Ultracef, Azactam, Videx, Zerit, Maxipime, VePesid, Paraplatin,Platinol, Taxol, UFT, Buspar, Serzone, Stadol NS, Estrace, Glucophage(Bristol-Myers Squibb);

[0097] (b) Ceclor, Lorabid, Dynabac, Prozac, Darvon, Permax, Zyprexa,Humalog, Axid, Gemzar, Evista (Eli Lily);

[0098] (c) Vasotec/Vaseretic, Mevacor, Zocor, Prinivil/Prinizide,Plendil, Cozaar/Hyzaar, Pepcid, Prilosec, Primaxin, Noroxin, RecombivaxHB, Varivax, Timoptic/XE, Trusopt, Proscar, Fosamax, Sinemet, Crixivan,Propecia, Vioxx, Singulair, Maxalt, Ivermectin (Merck & Co.);

[0099] (d) Diflucan, Unasyn, Sulperazon, Zithromax, Trovan, ProcardiaXL, Cardura, Norvasc, Dofetilide, Feldene, Zoloft, Zeldox, Glucotrol XL,Zyrtec, Eletriptan, Viagra, Droloxifene, Aricept, Lipitor (Pfizer);

[0100] (e) Vantin, Rescriptor, Vistide, Genotropin,Micronase/Glyn./Glyb., Fragmin, Total Medrol, Xanax/alprazolam, Sermion,Halcion/triazolam, Freedox, Dostinex, Edronax, Mirapex, Pharmorubicin,Adriamycin, Camptosar, Remisar, Depo-Provera, Caverject, Detrusitol,Estring, Healon, Xalatan, Rogaine (Pharmacia & Upjohn);

[0101] (f) Lopid, Accrupil, Dilantin, Cognex, Neurontin, Loestrin,Dilzem, Fempatch, Estrostep, Rezulin, Lipitor, Omnicef, FemHRT, Suramin,Clinafloxacin (Warner Lambert).

[0102] Further examples of therapeutic agents which may be delivered bythe FcRn binding partners of the present invention may be found in:Goodman and Gilman's The Pharmacological Basis of Therapeutics. 9th ed.McGraw-Hill 1996, incorporated herein by reference in its entirety.

[0103] 5.4. Pharmaceutical Formulations for Vaccine Delivery

[0104] When administered, the conjugates of the present invention areadministered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines, and optionally other therapeutic agents. Thus, “cocktails”including the conjugates and the agents are contemplated. The agentsthemselves may be conjugated to FcRn binding partners to enhancedelivery of the agents across the epithelial barriers.

[0105] The conjugates of the invention may be administered in a purifiedform or in the form of a pharmaceutically acceptable salt. When used inmedicine the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmaceutically acceptable saltsinclude, but are not limited to, those prepared from the followingacids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic,acetic, salicylic, p-toluene sulphonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, andbenzene sulphonic. Also, pharmaceutically acceptable salts can beprepared as alkyline metal or alkyline earth salts,/such as sodium,potassium or calcium salts of the carboxylic acid group.

[0106] Suitable buffering agents include: acetic-acid and salt (1-2%W/V); citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2:5,% WN); sodium bicarbonate (0.5-1.0% W/V); and phosphoric acid and a salt(0.8-2% W/V). Suitable preservatives include benzalkonium chloride(0.003-0.03% W/V); chlotubutanol (0.3-0.9% W/V); parabens (0.01-0.25%W/V) and thimerosal (0.004-0.02% W/V).

[0107] The term “carrier” as used herein, and described more fullybelow, means one or more solid or liquid filler, dilutants orencapsulating substances which are suitable for administration to ahuman or other mammal. The “carrier” may be an organic or inorganicingredient, natural or synthetic, with which the active ingredient iscombined to facilitate administration.

[0108] The components of the pharmaceutical compositions are capable ofbeing commingled with the conjugates of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy. The componentsof oral drug formulations include diluents, binders, lubricants,glidants, disintegrants, coloring agents and flavoring agents.Encapsulating substances for the preparation of enteric-coated oralformulations include cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate and methacrylic acidester copolymers. Solid oral formulations such as capsules or tabletsare preferred. Elixirs and syrups also are well known oral formulations.The components of aerosol formulations include solubilized activeingredients, antioxidants, solvent blends and propellants for solutionformulations, and micronized and suspended active ingredients,dispersing agents and propellants for suspension formulations. The oral,aerosol and nasal formulations of the invention can be distinguishedfrom injectable preparations of the prior art because such formulationsmay be nonaseptic, whereas injectable preparations must be aseptic.

[0109] The term “adjuvant” is intended to include any substance which isincorporated into or administered simultaneously with the conjugates ofthe invention and which nonspecifically potentiates the immune responsein the subject. Adjuvants include aluminum compounds, e.g., gels,aluminum hydroxide and aluminum phosphate, and Freund's complete orincomplete adjuvant (in which the conjugate is incorporated in theaqueous phase of a stabilized water in paraffin oil emulsion). Theparaffin oil may be replaced with different types of oils, e.g.,squalene or peanut oil. Other materials with adjuvant properties includeBCG (attenuated Mycobacterium tuberculosis), calcium phosphate,levamisole, isoprinosine, polyanions (e.g., poly A:U) leutinan,pertussis toxin, cholera toxin, lipid A, saponins and peptides, e.g.muramyl dipeptide. Rare earth salts, e.g., lanthanum and cerium, mayalso be used as adjuvants. The amount of adjuvants depends on thesubject and the particular conjugate used and can be readily determinedby one skilled in the art without undue experimentation.

[0110] Other supplementary immune potentiating agents, such ascytokines, may be delivered in conjunction with the conjugates of theinvention. The cytokines contemplated are those that will enhance thebeneficial effects that result from administering the immunomodulatorsaccording to the invention. Cytokines are factors that support thegrowth and maturation of cells, including lymphocytes. It is believedthat the addition of cytokines will augment cytokine activity stimulatedin vivo by carrying out the methods of the invention. The preferredcytokines are interleukin (IL)-1, IL-2, gamma-interferon and tumornecrosis factor α. Other useful cytokines are believed to be IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,erythropoietin, leukemia inhibitory factor, oncostatin-M, ciliaryneurotrophic factor, growth hormone, prolactin, CD40-ligand,CD27-ligand, CD30-ligand, alpha-interferon, beta-interferon, and tumornecrosis factor-β. Other cytokines known to modulate T-cell activity ina manner likely to be useful according to the invention are colonystimulating factors and growth factors including granulocyte and/ormacrophage stimulating factors (GM-CSF, G-CSF and CSF-1) and plateletderived, epidermal, insulin-like, transforming and fibroblast growthfactors. The selection of the particular cytokines will depend upon theparticular modulation of the immune system that is desired. The activityof cytokines on particular cell types is known to those of ordinaryskill in the art.

[0111] The precise amounts of the foregoing cytokines used in theinvention will depend upon a variety of factors, including the conjugateselected, the dose and dose-timing selected, the mode of administrationand the characteristics of the subject. The precise amounts selected canbe determined without undue experimentation, particularly since athreshold amount will be any amount which will enhance the desiredimmune response. Thus, it is believed that nanogram to milligram amountsare useful, depending upon the mode of delivery, but that nanogram tomicrogram amounts are likely to be most useful because physiologicallevels of cytokines are correspondingly low.

[0112] The preparations of the invention are administered in effectiveamounts. An effective amount is that amount of a conjugate that willalone, or together with further doses, stimulate an immune response asdesired. This may involve the stimulation of a humoral antibody responseresulting in an increase in antibody titer in serum, improved mucosalimmunity, a clonal expansion of cytotoxic T lymphocytes or tolerance toan antigen, including a self antigen. It is believed that doses rangingfrom 1 nanogram/kilogram to 100 milligrams/kilogram, depending upon themode of administration, will be effective. The preferred range isbelieved to be between about 500 nanograms and 500 micrograms/kilogram,and most preferably between 1 microgram and 100 micrograms/kilogram. Theabsolute amount will depend upon a variety of factors, including theconjugate selected, the immune modulation desired, whether theadministration is in a single or multiple doses, and individual patientparameters including age, physical condition, size and weight. Fortreatment of a subject with a tumor the size, type, location andmetastases of the tumor may be factored in when determining the amountof conjugate to administer. These factors are well known to those ofordinary skill in the art and can be addressed with no more than routineexperimentation.

[0113] A variety of administration routes are available. The particularmode selected will depend, of course, upon the particular conjugateselected, the particular condtion being treated and the dosage requiredfor therapeutic efficacy. The methods of this invention, generallyspeaking, involve delivering the conjugates of the invention to anepithelial surface. Preferred modes of administration are oral,intrapulmonary, intrabinary and intranasal.

[0114] Compositions may be conveniently presented in unit dosage formand may be prepared by any of the methods well known in the art ofpharmacy. All methods include the step of bringing the conjugate intoassociation with a carrier which constitutes one or more accessory,ingredients. In general, the compositions are prepared by uniformly andintimately bringing the conjugate into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product.

[0115] Other delivery systems can include time-release, delayed releaseor sustained release delivery systems. Such systems can avoid repeatedadministrations of the conjugates of the invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer based systems such as polytactic andpolyglycolic acid, polyanhidrides and polycaprolactone; wax coatings,compressed tablets using conventional binders and excipients, and thelike. Bioadhesive polymer systems to enhance delivery of a material tothe intestinal epithelium are known and described in published PCTapplication WO 93/21906. Capsules for delivering agents to theintestinal epithelium also are described in published PCT application WO93/19660.

[0116] 5.5. Pharmaceutical Formulations for Administration ofTherapeutics

[0117] The pharmaceutical formulation of the invention contain the FcRnbinding partner conjugate as the active ingredient in a pharmaceuticallyacceptable carrier suitable for administration and delivery in vivo. Inpreferred embodiments the pharmaceutical compositions of the presentinvention are formulated for oral, sublingual, buccal, intranasal andadministration by inhalation.

[0118] When administered, the conjugates of the present invention areadministered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines, and optionally other therapeutic agents. Thus, “cocktails”including the conjugates and the agents are contemplated. The agentsthemselves may be conjugated to FcRn binding partners to enhancedelivery of the agents across the epithelial barriers.

[0119] 5.5.1. Dosages

[0120] The preferred amount of FcRn binding partner conjugates in allpharmaceutical preparations made in accordance with the presentinvention should be a therapeutically effective amount thereof which isalso a medically acceptable amount thereof. Actual dosage levels of FcRnbinding partner conjugates in the pharmaceutical compositions of thepresent invention may be varied so as to obtain an amount of FcRnbinding partner conjugates which is effective to achieve the desiredtherapeutic response for a particular patient, pharmaceuticalcomposition of FcRn binding partner conjugates, and mode ofadministration, without being toxic to the patient.

[0121] The selected dosage level and frequency of administration willdepend upon a variety of factors including the route of administration,the time of administration, the rate of excretion of the therapeuticagent(s) including FcRn binding partner conjugates, the duration of thetreatment, other drugs, compounds and/or materials used in combinationwith FcRn binding partner conjugates, the age, sex, weight, condition,general health and prior medical history of the patient being treatedand the like factors well known in the medical arts. For example, thedosage regimen is likely to vary with pregnant women, nursing mothersand children relative to healthy adults.

[0122] A physician having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician couldstart doses of FcRn binding partner conjugates employed in thepharmaceutical composition of the present invention at levels lower thanthat required to achieve the desired therapeutic effect and graduallyincrease the dosage until the desired effect is achieved.

[0123] 5.5.2. Pharmaceutical Formulations

[0124] The pharmaceutical compositions of the present invention,including the FcRn binding partners conjugated to a therapeutic as theactive agent are suitable preferably for oral, sublingual, andintranasal delivery. The pharmaceutical compositions are suitable forthe delivery of the FcRn binding partner conjugates to epithelialbarriers. The pharmaceutical compositions may also be formulated to besuitable for parenteral, transdermal, intradermal and intravenousdelivery.

[0125] The pharmaceutical compositions, containing biologically activeFcRn binding partner conjugates as the active agent, that are suitablefor transmucosal delivery via oral cavity delivery are in the form of asolid as lingual, buccal or sublingual tablets, troches (lozenges),powders, time-release granules, pellets or the like may also be used, orin the form of a liquid as a liquid drop or drops, aerosol spray ormist, applied sublingually (under the tongue), on top of the tongue, orbuccally (between the cheek and gingiva). The rate of oral mucosalmembrane absorption of FcRn binding partner conjugates, is controlled bythe specific liquid or solid dosage formulation selected. Specificformulations allow the process of absorption to take place over asustained, but relatively short period of time, allowing for a gradualbuild up and constant blood level of the FcRn binding partnerconjugates.

[0126] For prolonged delivery, the active ingredient can be formulatedas a depot preparation, for administration by implantation; e.g.,subcutaneous, intradermal, or intramuscular injection. Thus, forexample, the active ingredient may be formulated with suitable polymericor hydrophobic materials (e.g., as an emulsion in an acceptable oil) orion exchange resins, or as sparingly soluble derivatives; e.g., as asparingly soluble salt form of the FcRn binding partner conjugate.

[0127] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated to give controlled release ofthe active compound. By way of example, but not by limitation, the FcRnbinding partners may be conjugated to the following therapeutics forepithelial barrier targeted delivery:

[0128] For buccal or sublingual administration, the compositions maytake the form of tablets or lozenges formulated in conventional manner.For rectal and vaginal routes of administration, the active ingredientmay be formulated as solutions (for retention enemas) suppositories orointments.

[0129] For administration by inhalation, the active ingredient can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

[0130] A variety of administration routes are available. The particularmode selected will depend, of course, upon the particular conjugateselected, the particular condtion being treated and the dosage requiredfor therapeutic efficacy. The methods of this invention, generallyspeaking, involve delivering the conjugates of the invention to anepithelial surface. Preferred modes of administration are oral,intrapulmonary, intrabinary and intranasal.

[0131] Compositions may be conveniently presented in unit dosage formand may be prepared by any of the methods well known in the art ofpharmacy. All methods include the step of bringing the conjugate intoassociation with a carrier which constitutes one or more accessory,ingredients. In general, the compositions are prepared by uniformly andintimately bringing the conjugate into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product.

[0132] Other delivery systems can include time-release, delayed releaseor sustained release delivery systems. Such systems can avoid repeatedadministrations of the conjugates of the invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer based systems such as polytactic andpolyglycolic acid, polyanhidrides and polycaprolactone; wax coatings,compressed tablets using conventional binders and excipients, and thelike. Bioadhesive polymer systems to enhance delivery of a material tothe intestinal epithelium are known and described in published PCTapplication WO 93/21906. Capsules for delivering agents to theintestinal epithelium also are described in published PCT application WO93/19660.

[0133] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

6. EXAMPLES

[0134] Materials

[0135] Abbreviations

[0136] BSA, bovine serum albumin; CDNA, complementary deoxyribonucleicacid; CT-8, cholera toxin 6 subunit; DMEM, Dulbecco's modified Eagle'smedium; DMSO, dimethyl sulfoxide; DOC, desoxycholate; ZCL, enhancedchemiluminescene, ELISA, enzyme linked immunosorbant assay; HBSS, Hanks'balanced salt solution without calcium or magnesium; HEPES,N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]; hGH, humangrowth hormone; IEC, intestinal epithelial cells; KI, potassium iodide;MHC, major histocompatibility complex; NAOH, sodium hydroxide; NH, CL,ammonium chloride; NHS-rhodamine, N-hydroxysuccinimidyl-rhodamine; RNA,ribonucleic acid; RT-PCR, reverse transcriptase-polymerase chainreaction; SATA, N-succinimdyl S-acetylthioacetate; SDS-PAGE, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis; sulfo-LC-SPDP,sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamide]hexanoate;sulfo-NHS-biotin, sulfosuccinimidobiotin; sulfo-SMCC, sulfosuccinimidyl4-(N-maleimidoriethyl) cyclo-hexane-1-carboxylate.

[0137] Chemicals

[0138] cDNA Cycle Kits was purchased from Invitrogen (San Diego,Calif.). TAO polymerase was purchased from Perkin-Elmer Cetus (Norwalk,Conn.). CircumVent™ Kits were purchased from New England Biolabs(Beverly, Mass.). Radionucleotides and radioactive chemicals werepurchased from DuPont/NEN (Boston, Mass.). HBSS and DMEM were purchasedfrom GIBCO/Life Technologies (Gaithersburg, Md.). RPMI 1640 waspurchased from Cellgro (Herndon, Va.). L-glutamine was purchased fromCellgro. Protein A-Sepharose was purchased from Pharmacia Biotech(Piscataway, N.J.). Streptavidin-horseradish peroxidase, sulfo-LC-SPDP,sulfo-NHS-biotin, sulfo-SMCC, SATA and immobilized ficin were purchasedfrom Pierce (Rockford, Ill.). Balb/c mice were purchased from CharlesRiver Laboratories (Wilmington, Mass.). ECL kits were purchased fromAmersham (Arlington Heights, Ill.). Plasmin, AvidChrom-protein A,protein G-Sepharose, BSA, cholera toxin B subunit, anti-hGH antibodiesand all other chemicals were purchased from Sigma (St. Louis, Mo.).

Example 1 Expression of FcRn mRNA in Human Intestinal Epithelial PrimaryCells and Cell Lines

[0139] Total RNA was extracted from adult human enterocytes by standardmethodology well known in the art (Sambrook et al., ibid.). Onemicrogram of RNA from each cell type was used as a template to preparethe cDNA substrate for reverse transcriptase-polymerase chain reaction(RT-PCR) using a CDNA Cycle Kit (invitrogen, San Diego, Calif.). Thirtycycles of PCR were performed on the cDNA using Taq polymerase(Perkin-Elmer Cetus, Norwalk, Conn.) according to the manufacturer'sinstructions using primers TGCTGGGCTGTGAACTG and CGCTTTTAGCAGTCGGAA. ThePCR cycle conditions were: denaturation at 94° C. for one minute,annealing at 55° C. for two minutes and extension at 72° C. for threeminutes. Amplification products were resolved by electrophoresis on a1.5% agarose gel and visualized by ethidium bromide staining, whichshowed the presence of the expected approximately 800 base pairamplification product in all samples except the adult colonic epithelialcells. To confirm the identity of the RT-PCR amplification product, theDNA band was excised from the agarose gel, subcloned into pCR 11(Invitrogen, San Diego, Calif.) and sequenced using a Prismdye-deoxyterminator cycle sequencing kit (Applied Biosystems, Foster City,Calif.) using primers from both vector and human FcRn sequence. Reactionproducts were analyzed on an Applied Biosystems sequencer. The sequenceof the amplification products exactly matched the FcRn gene sequence,confirming the identity of the expressed gene.

Example 2 Detection of FcRn mRNA by Northern Blot

[0140] To confirm the expression of FcRn in human intestinal epithelialcells and cell lines, a Northern blot was prepared using the RNA samplesprepared as described in Example 1 from adult human enterocytes, andfrom two human adenocarcinoma cell lines of colonic origin, CaCO-2 andHT-29. The RNA samples were resolved by formaldehyde/agarose gelelectrophoresis and transferred to a nylon membrane by standardprocedures (Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1989). The membranewas probed using a ³²P-radiolabeled 120 base pair probe from the 3′untranslated region of FcRn by standard methods. Autoradiograms of theNorthern blot demonstrated the presence of the 1.5 kilobase hFcRntranscript in the enterocytes and both cell lines. Therefore, theexpression of FcRn in human adult intestinal epithelial cells and celllines was demonstrated by two different methods of RNA detection.

Example 3 Labeling and Immunoprecipitation of the MHC-Class I Related FcReceptor (FcRn) from Intestinal Epithelial Cells

[0141] The expression of FcRn in human intestinal epithelial cells wasconfirmed by immunoprecipitation of the protein. Caco-2 cells werelabeled metabolically using IIS-methionine (DuPont/NEN, Boston, Mass.)and proteins were extracted by methods well known in the art (Harlow andLane, Antibodies: A Laboratory Manual). A polyclonal rabbit anti-rat MHCclass I related FcR heavy chain specific antiserum bound toprotein-A-sephorose was used to immunoprecipitate FcRn from the cellextracts using standard methods (FcRn can be purified by wellestablished methods, Simister and Rees 1985, European J. Immunology,15:733-8, and used to Immunize rats followed by collection of serum,Harlow and Lane, supra.). Immunoprecipitates were resolved by SDS-PAGEand visualized by autoradiography. A 48 kilodalton FcRn protein wasobserved, confirming expression observed at the RNA level.

Example 4 Expression of FcRn Protein on the Cell Surface of HumanIntestinal Epithelial Cells

[0142] About 3×10⁷ HT-29 intestinal epithelial cells were detached fromtissue culture plates by nonenzymatic methods and were washed four timeswith ice cold Hanks' balanced salt solution containing no calcium ormagnesium (HBSS-, GIBCO/Life Technologies, Gaithersburg, Md.). To labelcell surface proteins, the washed cells were incubated twice for 20minutes with 1.5 mi of 0.5 mg/mi sulfo-NHS-biotin (Pierce, Rockford,Ill.) in DMSO. Labeled cells were washed five times with 50 mM NH₄C,incubated 20 minutes with 1 0 ml of RPMI 1640 (Cellgro, City, State)containing 1 mM L-glutamine (Mediatech, Washington, D.C.), and washedfour times with HBSS-. The cells were lysed, then precleared overnightwith protein A-Sepharose beads (Pharmacia Biotech, Piscataway, N.J.)using standard techniques well known in the art. SDS and desoxycholicacid (DOC) were added to the supernatants to final concentrations of0.1% and 0.5%, respectively. Lysates were precleared with normal rabbitserum and immunoprecipitated with polyclonal rabbit anti-rat MHC class Irelated FcR antibody by methods well known in the art.Immunoprecipitates were resolved by SDS-PAGE and transferred tonitrocellulose membranes. The nitrocellulose membrane was processed forincubation with 1:10,000 diluted streptavidinhorseradish peroxidase(Pierce, Rockford Ill.) as recommended by the manufacturer. The membranewas then processed for detection of bound horseradish peroxidase usingan ECL kit (Amersham, Arlington Heights, Ill.). Light emitted bycleavage of the chemiluminescent substrate was detected by exposure ofthe membrane to light-sensitive film. The film exposures showed thatFcRn was expressed on the surface of HT-29 intestinal epithelial cells.

Example 5 Functional Activity of Human FcRn on the Cell Surface ofIntestinal Epithelial Cells

[0143] To show that the FcRn expressed on the cell surface of intestinalepithelial cells was functional, Caco-2 cells and human adult jejunalintestinal epithelial cells (IECs) were tested for the ability to bindFc fragment of antibody. Caco-2 and jejunal I ECs were distributed tomicrocentrifuge tubes (2×10⁶ cells per tube) and pelleted at 2000 rpmfor 2-3 minutes at 4° C. Cell pellets were washed once in DMEMcontaining 20 mM HEPES, pH 6.0 or pH 8.0 at 4° C. and resuspended in 0.2ml of the same medium. The cell suspensions were transferred to 12 wellplates for assay. ¹²⁵1-Fc fragment (200 ng/ml, 4×10⁻⁹ M) in DMEMcontaining 20 mM HEPES, 1.0 mM Ki and 0.1% fish gelatin, pH 6.0 or pH8.0 with or without 0.5 mg/ml unlabeled human IgG (3.3×1 O-6M) was addedto each well. The cells were allowed to bind IgG or Fc at 37° C. for twohours in a 5% CO₂ humidified atmosphere. Cells were transferred tomicrocentrifuge tubes and pelleted at 2000 rpm for 2-3 minutes at 4° C.Unbound ¹²⁵I-Fc was removed by washing the cell pellets once with coldDMEM containing 20 mM HEPES, pH 6.0 or pH 8.0 at 4° C. Cells weredisrupted in 0.5 ml 0.1 M NAOH and the resulting solution transferred toscintillation vials. ¹²⁵I was quantified using a CliniGamma 1272 gammacounter (LKB Wallac, Piscataway, N.J.). Both Caco-2 cells and humanadult jejunum IECs specifically bound ¹²⁵I-Fc at pH 6.0 but not at pH8.0, demonstrating functional pH-dependent binding as observed for ratneonatal FcRn and cloned human FcRn (Story et al., J. Exp. Med. 180:2377-2381; December 1994).

Example 6 Preparation of Human Immunoglobulin G

[0144] Non-specific purified immunoglobulin G from human, mouse, rat,goat, pig, cow, and other species may be purchased from commercialvendors such as Sigma Chemical Co., Pierce Chemical, HyCloneLaboratories, ICN Biomedicals and Organon Teknika-Cappei.

[0145] Immunoglobulin G also may be isolated by ammonium sulfateprecipitation of precipitation of blood serum. The protein precipitateis further fractionated by ion exchange chromatography or gel filtrationchromatography, to isolate substantially purified non-specific IgG. Bynon-specific IgG it is meant chat no single specificity within theantibody population or pool is dominant.

[0146] Immunoglobulin G also may be purified from blood serum byadsorption to protein A attached to a solid support such as proteinA-Sepharose (Pharmacia), AvidChrom-Protein A (Sigma), or proteinG-Sepharose (Sigma). Other methods of purification of IgG are well knownto persons skilled in the art and may be used for the purpose ofisolation of non-specific IgG.

Example 7 Preparation of Human Lmmuglobulin G

[0147] To prepare the Fc fragments of human IgG, IgG isolated as inexample 6 is subjected to digestion with immobilized papain (Pierce)according to the manufacturer's recommended protocol. Other proteasesthat digest IgG to produce intact Fc fragments that can bind to Fcreceptors, e.g. plasmin (Sigma) or immobilized ficin (Pierce), are knownto skilled artisans and may be used to prepare Fc fragments. Thedigested immunoglobulin then is incubated with an affinity matrix suchas protein A-Sepharose or protein G-Sepharose. Non-binding portions ofIgG are eluted from the affinity matrix by extensive washing in batch orcolumn format. Fc fragments of IgG then are eluted by addition of abuffer that is incompatible with Fc-adsorbent binding. Othermethodologies effective in the purification of Fc fragments also may beemployed.

Example 8 Conjugation of Compounds to Human Immunoglobulin Fc-Fragments

[0148] To deliver compounds via the FcRn transport mechanism, suchcompounds can be coupled to whole IgG or Fc fragments. The chemistry ofcross-linking and effective reagents for such purposes are well known inthe art. The nature of the crosslinking reagent used to conjugate wholeIgG or Fc fragments and the compound to be delivered is not restrictedby the invention. Any crosslinking agent may be used provided that a)the activity of the compound is retained, and b) binding by the FcRn ofthe Fc portion of the conjugate is not adversely affected.

[0149] An example of an effective one-step crosslinking of Fc and acompound is oxidation of Fc with sodium periodate in sodium phosphatebuffer for 30 minutes at room temperature, followed by overnightincubation at 4° C. with the compound to be conjugated. Conjugation alsomay be performed by derivatizing both the compound and Fc fragments withsuffosuccinimidyl 6-[3-(2-pyridyldithio)propionamidel hexanoate(sulfo-LC-SPDP, Pierce) for 18 hours at room temperature. Conjugatesalso may be prepared by derivatizing Fc fragments and the desiredcompound to be delivered with different crosslinking reagents that willsubsequently form a covalent linkage. An example of this reaction isderivatization of Fc fragments with sulfosuccinimidyl4-(N-maleimidomethyl) cyclo-hexane-1-carboxylate (Sulfo-SMCC, Pierce)and the compound to be conjugated to Fc is thiolated with N-succinimidylS-acetylthioacetate (SATA). The derivatized components are purified freeof crosslinker and combined at room temperature for one hour to allowcrosslinking. Other crosslinking reagents comprising aldehyde, imide,cyano, halogen, carboxyl, activated carboxyl, anhydride and maleimidefunctional groups are known to persons of ordinary skill in the art andalso may be used for conjugation of compounds to Fc fragments. Thechoice of cross-linking reagent will, of course, depend on the nature ofthe compound desired to be conjugated to Fc. The crosslinking reagentsdescribed above are effective for protein-protein conjugations. If thecompound to be conjugated is a carbohydrate or has a carbohydratemoiety, then heterobifunctional crosslinking reagents such as ABH,M2C2H, MPBH and PDPH are useful for conjugation with a proteinaceousFcRn binding molecule (Pierce Chemical Co., Rockford, Ill.). Anothermethod of conjugating proteins and carbohydrates is disclosed byBrumeanu et al. (Genetic Engineering News, Oct. 1, 1995, p. 16). If thecompound to be conjugated is a lipid or has a lipid moiety which isconvenient as a site of conjugation for the FcRn binding molecule, thencrosslinkers such as SPDP, SMPB and derivatives thereof may be used(Pierce Chemical Co., Rockford, Ill.). It is also possible to conjugateany molecule which is to be delivered by noncovalent means. Oneconvenient way for achieving noncovalent conjugation is to raiseantibodies to the compound to be delivered, such as monoclonalantibodies, by methods well known in the art, and select a monoclonalantibody having the correct Fc region and desired antigen bindingproperties. The antigen to be delivered is then prebound to themonoclonal antibody carrier. In all of the above crosslinking reactionsit is important to purify the derivatized compounds free of crosslinkingreagent. It is important also to purify the final conjugatesubstantially free of unconjugated reactants. Purification may beachieved by affinity, gel filtration or ion exchange chromatographybased on the properties of either component of the conjugate. Aparticularly preferred method is an initial affinity purification stepusing protein A-Sepharose to retain Fc and Fc-compound conjugates,followed by gel filtration or ion exchange chromatography based on themass, size or charge of the Fc conjugate. The initial step of thispurification scheme ensures that the conjugate will bind to FcRn whichis an essential requirement of the invention.

[0150] Example 9

IgG-Facilitated Delivery of Foreign Antigen Across the IntestinalEpithelial Barrier

[0151] To test the ability of Fc binding partner-antigen conjugates tobe transported across epithelial barriers, foreign antigens areconjugated to IgG molecules for administration to mice. A convenientforeign antigen is the fluorescent dye rhodamine, since it may bevisualized in frozen semi-thin sections of intestinal epithelium.Rhodamine is covalently linked to non-specific mouse IgG, prepared asdescribed in Example 6, cholera toxin B subunit (Sigma) and ovalbumin(Sigma) by incubation with succinyl-rhodamine (Molecular Probes, Eugene,Oreg.) as recommended by the manufacturer. The IgG-rhodamine conjugateis purified by protein G-Sepharose affinity chromatography. Afterdialysis to remove unconjugated succinyl-rhodamine, cholera toxin B(CT-B)-rhodamine and ovalbumin-rhodamine conjugates are purified by gelfiltrations or ion exchange chromatography. Fractions of the eluate areassayed for the presence of conjugates by determining fluorescence.Functional binding of the IgG and CT-B subunit conjugates may be testedby binding to FcRn and ganglioside GM1, respectively. Cholera toxinB-rhodamine and ovalbuminrhodamine serve as positive and negativecontrols, respectively.

[0152] Balb/c mice are administered 0.2 nanomoles of the three rhodamineconjugates described above, with or without 0.2 nanomoles unlabeledcholera toxin as a non-specific adjuvant, by intragastric administrationin the presence of 75 micromoles NaHCO₃ and 20 mg/mi soybean trypsininhibitor to inhibit gastric degradation. After 6 hours the mice aresacrificed and intestine is removed, frozen and processed for semi-thinsectioning. Sections of the intestinal epithelium are illuminated on afluorescent microscope and examined for intracellular fluorescence. Thepresence of fluorescence in intestinal epithelial cells of mice fedIgG-rhodamine indicates that the IgG conjugates are effectivelytransported in an apical to basolateral direction across the intestinalepithelial barrier. FcRn is capable of transporting immunogens asconjugates with FcRn binding partners.

Example 10 Mouse Mucosal Immune Response to Orally Delivered Antigen-IgGConjugate via FcRn-Mediated Transcytosis

[0153] Transgenic mice homozygous for deletion of β2-microglobulin (acritical component of Fc-receptor function) and their normal wild-typelitter mates are used for studies of generation of a mucosal immuneresponse. If rhodamine-IgG elicits a mucosal immune response by bindingto apical membrane Fc receptors, a positive immune response should befound in wild-type but not β2-microglobulin “knockout” mice. Incontrast, rhodamine-cholera toxin B subunit (CT-6) should elicit apositive immune response in both wild type and—knockout” mice astranscytosis of CT-6 across the epithelial barrier is not dependent onbinding to apical membrane Fc receptors. Rhodamine-ovalbumin does, notenter transcytotic vesicles (but may enter intestinal epithelia by fluidphase endocytosis) and should not elicit an immune response in any mice.

[0154] Three groups of wild type and β₂-microglobulin knockout mice areorally immunized with the three rhodamine conjugates described inExample 9. Parallel experiments are conducted with the addition of 0.2nanomoles of cholera toxin as non-specific adjuvant. Equimolarquantities of the rhodamine conjugates are administeredintragastrically. The mice are “immunized” by this method every ten daysfor a total of three times. Two weeks after the third oral immunizationthe mice are sacrificed and the rhodamine-specific immune response isdetermined by ELISA on gut secretions and serum by standard methodology.Anti-rhodamine serum immunoglobulins are most evident in the wild typemice fed rhodamine conjugates of CT-B and IgG. Knockout mice lacking112-microglobulin generate a mucosal immune response to rhodamine-CT-Bbut not to rhodamine-IgG, indicating that receptor-mediated transcytosisplays an essential role in the mucosal immune response. The controlrhodamine-ovalbumin conjugate elicits little or no immune response ineither the wild type or the β₂-Microglobulin knockout mice.

[0155] Those skilled in the art will be able to recognize or ascertainwith no more than routine experimentation numerous equivalents to thespecific products and processes described above. Such equivalents areconsidered to be within the scope of the invention and are intended tobe covered by the following claims in which we claim:

Example 11 IgG-Facilitated Delivery of the Bioactive Substance Insulin,Across the Intestinal Epithelial Barrier

[0156] To test the ability of Fc binding partner-bioactive substanceconjugates to be transported across epithelial barriers, the bioactivesubstance, insulin is conjugated to IgG molecules for administration tomice. A convenient therapeutic agent to be delivered is insulin, as itspresence in systemic circulation can be determined by measuring adecrease in blood glucose levels. The results of such an assaydemonstrate the efficacy of the FcRn binding partner delivery system.Insulin is covalently linked to non-specific mouse IgG, prepared asdescribed in Example 6, cholera toxin B subunit (Sigma) and ovalbumin(Sigma) by incubation with succinyl-insulin (Molecular Probes, Eugene,Oreg.) as recommended by the manufacturer. The IgG-insulin conjugate ispurified by protein G-Sepharose affinity chromatography. After dialysisto remove unconjugated succinyl-Insulin, cholera toxin B (CT-B)-Insulinand ovalbumin-Insulin conjugates are purified by gel filtrations or ionexchange chromatography. Fractions of the eluate are assayed for thepresence of conjugates by determining fluorescence. Functional bindingof the IgG and CT-B subunit conjugates may be tested by binding to FcRnand ganglioside GM1, respectively. Cholera toxin B-Insulin and ovalbuminInsulin serve as positive and negative controls, respectively.

[0157] Balb/c mice are administered 0.2 nanomoles of the three Insulincojugates described above, with or without 0.2 nanomoles unlabeledcholera toxin as a non-specific control, by oral administration in thepresence of 75 micromoles NaHCO₃ and 20 mg/mi soybean trypsin inhibitorto inhibit gastric degradation.

[0158] In vivo Insulin Delivery

[0159] Balb/C mice of female sex were fasted for 12 hours beforeexperiment. Each mouse was gavaged 200 μL of each preparation asdescribed in Table 9.3. Food was restored immediately afteradministration. Blood samples for glucose determination was drawn frommouse tail vein under methoxyflurane anesthesia. Samples were drawnright before the administration (0 hour), as well as at 1, 2, 3 and 3.5hours post administration of each preparation. Blood glucose level wasmeasured using a One Touch® Profile Diabetes Tracking System (Lifescan,Milpitas, Calif.) with one Touch® Test strips, by applying blood to forma round drop which completely covered the test spot on the test strip.Readings (in mg/dL) were obtained from the meter indicating the bloodglucose level detected.

[0160] The invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

[0161] All references cited herein are incorporated herein by referencefor all purposes.

1 22 1 706 DNA Homo sapiens 1 gtcgtcgaca aaactcacac atgcccaccgtgcccagcac ctgaactcct ggggggaccg 60 tcagtcttcc tcttcccccc aaaacccaaggacaccctca tgatctcccg gacccctgag 120 gtcacatgcg tggtggtgga cgtgagccacgaagaccctg aggtcaagtt caactggtac 180 gtggacggcg tggaggtgca taatgccaagacaaagccgc gggaggagca gtacaacagc 240 acgtaccgtg tggtcagcgt cctcaccgtcctgcaccagg actggctgaa tggcaaggag 300 tacaagtgca aggtctccaa caaagccctcccagccccca tcgagaaaac catctccaaa 360 gccaaagggc agccccgaga accacaggtgtacaccctgc ccccatcccg ggatgagctg 420 accaagaacc aggtcagcct gacctgcctggtcaaaggct tctatcccag cgacatcgcc 480 gtggagtggg agagcaatgg gcagccggagaacaactaca agaccacgcc tcccgtgttg 540 gactccgacg gctccttctt cctctacagcaagctcaccg tggacaagag caggtggcag 600 caggggaacg tcttctcatg ctccgtgatgcatgaggctc tgcacaacca ctacacgcag 660 aagagcctct ccctgtctcc gggtaaatgagtgcggcggc cgcgtc 706 2 229 PRT Homo sapiens 2 Val Val Asp Lys Thr HisThr Cys Pro Pro Cys Pro Ala Pro Glu Leu 1 5 10 15 Leu Gly Gly Pro SerVal Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser ArgThr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser His Glu Asp ProGlu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn AlaLys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 65 70 75 80 Thr Tyr Arg ValVal Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys GluTyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 100 105 110 Pro Ile GluLys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln ValTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 130 135 140 ValSer Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser CysSer 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys SerLeu Ser 210 215 220 Leu Ser Pro Gly Lys 225 3 5 PRT Homo sapiens 3 ValArg Arg Pro Arg 1 5 4 33 PRT Homo sapiens 4 Ser Ser Thr Lys Leu Thr HisAla His Arg Ala Gln His Leu Asn Ser 1 5 10 15 Trp Gly Asp Arg Gln SerSer Ser Ser Pro Gln Asn Pro Arg Thr Pro 20 25 30 Ser 5 13 PRT Homosapiens 5 Ser Pro Gly Pro Leu Arg Ser His Ala Trp Trp Trp Thr 1 5 10 647 PRT Homo sapiens 6 Ala Thr Lys Thr Leu Arg Ser Ser Ser Thr Gly ThrTrp Thr Ala Trp 1 5 10 15 Arg Cys Ile Met Pro Arg Gln Ser Arg Gly ArgSer Ser Thr Thr Ala 20 25 30 Arg Thr Val Trp Ser Ala Ser Ser Pro Ser CysThr Arg Thr Gly 35 40 45 7 43 PRT Homo sapiens 7 Met Ala Arg Ser Thr SerAla Arg Ser Pro Thr Lys Pro Ser Gln Pro 1 5 10 15 Pro Ser Arg Lys ProSer Pro Lys Pro Lys Gly Ser Pro Glu Asn His 20 25 30 Arg Cys Thr Pro CysPro His Pro Gly Met Ser 35 40 8 6 PRT Homo sapiens 8 Pro Arg Thr Arg SerAla 1 5 9 61 PRT Homo sapiens 9 Pro Ala Trp Ser Lys Ala Ser Ile Pro AlaThr Ser Pro Trp Ser Gly 1 5 10 15 Arg Ala Met Gly Ser Arg Arg Thr ThrThr Arg Pro Arg Leu Pro Cys 20 25 30 Trp Thr Pro Thr Ala Pro Ser Ser SerThr Ala Ser Ser Pro Trp Thr 35 40 45 Arg Ala Gly Gly Ser Arg Gly Thr SerSer His Ala Pro 50 55 60 10 26 PRT Homo sapiens 10 Cys Met Arg Leu CysThr Thr Thr Thr Arg Arg Arg Ala Ser Pro Cys 1 5 10 15 Leu Arg Val AsnGlu Cys Gly Gly Arg Val 20 25 11 13 PRT Homo sapiens 11 Arg Arg Gln AsnSer His Met Pro Thr Val Pro Ser Thr 1 5 10 12 24 PRT Homo sapiens 12 ThrPro Gly Gly Thr Val Ser Leu Pro Leu Pro Pro Lys Thr Gln Gly 1 5 10 15His Pro His Asp Leu Pro Asp Pro 20 13 13 PRT Homo sapiens 13 Gly His MetArg Gly Gly Gly Arg Glu Pro Arg Arg Pro 1 5 10 14 13 PRT Homo sapiens 14Gly Gln Val Gln Leu Val Arg Gly Arg Arg Gly Gly Ala 1 5 10 15 70 PRTHomo sapiens 15 Cys Gln Asp Lys Ala Ala Gly Gly Ala Val Gln Gln His ValPro Cys 1 5 10 15 Gly Gln Arg Pro His Arg Pro Ala Pro Gly Leu Ala GluTrp Gln Gly 20 25 30 Val Gln Val Gln Gly Leu Gln Gln Ser Pro Pro Ser ProHis Arg Glu 35 40 45 Asn His Leu Gln Ser Gln Arg Ala Ala Pro Arg Thr ThrGly Val His 50 55 60 Pro Ala Pro Ile Pro Gly 65 70 16 72 PRT Homosapiens 16 Ala Asp Gln Glu Pro Gly Gln Pro Asp Leu Pro Gly Gln Arg LeuLeu 1 5 10 15 Ser Gln Arg His Arg Arg Gly Val Gly Glu Gln Trp Ala AlaGly Glu 20 25 30 Gln Leu Gln Asp His Ala Ser Arg Val Gly Leu Arg Arg LeuLeu Leu 35 40 45 Pro Leu Gln Gln Ala His Arg Gly Gln Glu Gln Val Ala AlaGly Glu 50 55 60 Arg Leu Leu Met Leu Arg Asp Ala 65 70 17 16 PRT Homosapiens 17 Gly Ser Ala Gln Pro Leu His Ala Glu Glu Pro Leu Pro Val SerGly 1 5 10 15 18 6 PRT Homo sapiens 18 Met Ser Ala Ala Ala Ala 1 5 19 17DNA Homo sapiens 19 tgctgggctg tgaactg 17 20 18 DNA Homo sapiens 20cgcttttagc agtcggaa 18 21 4 PRT Homo sapiens 21 Asp Lys Thr His 1 22 4PRT Homo sapiens 22 Ser Pro Gly Lys 1

1. A method for modulating the immune system of a mammal, comprisingadministering to an epithelial barrier of a mammal in need of suchimmune modulation an effective amount of a conjugate of an antigen and aFcRn binding partner, wherein the antigen is selected from the groupconsisting of: an antigen that is characteristic of a pathogen, anantigen that is characteristic of an autoimmune disease, an antigen thatis characteristic of an allergen, and an antigen that is characteristicof a tumor.
 2. The method of claim 1 wherein the conjugate isadministered orally to the intestinal epithelium.
 3. The method of claim1 wherein the conjugate is administered in an aerosol to the lungs. 4.The method of claim 1 wherein the FcRn binding partner is non-specificIgG or a FcRn binding fragment of IgG.
 5. The method of claim 1 whereinthe FcRn binding partner is a Fc fragment of IgG.
 6. The method ofclaims 1-5 wherein the antigen is covalently coupled to the FcRn bindingpartner.
 7. The method of claims 1-5 wherein the antigen ischaracteristic of the tumor.
 8. The method of claim 7 wherein theantigen is covalently coupled to the FcRn binding partner.
 9. The methodof claims 1-5 wherein the antigen that is characteristic of the tumor isselected from the group consisting of proteins encoded by mutatedoncogenes; viral proteins associated with tumors; and tumor mucins andglycolipids.
 10. A pharmaceutical preparation comprising a conjugate ofan antigen and a FcRn binding partner, wherein the antigen ischaracteristic of a tumor, and a pharmaceutically acceptable carrier,wherein the conjugate is present in an amount effective for modulatingthe immune response of a mammal.
 11. The pharmaceutical preparation ofclaim 10 wherein the FcRn binding partner is non-specific IgG or a FcRnbinding fragment of IgG.
 12. The pharmaceutical preparation of claim 10wherein the FcRn binding partner is an Fc fragment of IgG.
 13. Thepharmaceutical preparation of claims 10-12 wherein the antigen iscovalently coupled to the FcRn binding partner.
 14. The pharmaceuticalpreparation of claims 10-12 wherein the unit dosage is an oralformulation.
 15. The pharmaceutical preparation of claims 10-12 whereinthe unit dosage is an aerosol formulations.
 16. The pharmaceuticalpreparation of claims 10-12 wherein the unit dosage is a nasalformulation.
 17. The pharmaceutical preparation of claim 10-12 whereinthe unit dosage is nonaseptic.
 18. A method of orally deliveringmolecules to a mammal which comprises administering to said mammal aneffective amount of a conjugate of a therapeutic and a FcRn bindingpartner targeted to epithelial cells expressing a FcRn.
 19. A method ofdelivering molecules to a mammal which comprises administering to saidmammal an effective amount of a conjugate of a bioactive substance and aFcRn binding partner targeted to epithelial cells expressing a FcRn. 20.The method of claim 19 wherein the FcRn binding partner is a Fc fragmentof IgG.
 21. The method of claim 19 wherein the method of delivery isoral or sublingual.
 22. The method of claim 19 wherein the method ofdelivery is intranasal.
 23. The method of claim 19 wherein the method ofdelivery is by aerosol administration.
 24. The method of claim 19wherein the bioactive substance is selected from the group consisting ofcells, viruses, vectors, proteins, peptides, nucleic acids,polysaccharides and carbohydrates, lipids, glycoproteins, andcombinations thereof and synthetic organic and inorganic drugs exertinga biological effect when administered to a mammal.